Glial cells are major constituents of the nervous system, and an array of devastating diseases, such as childhood periventricular leukomalacia, Alexanders Disease, and demyelinating diseases such as multiple sclerosis, arise from glial malfunction. Although glial cells have been recently recognized as playing key roles in neuronal function, sculpting synaptic connections, and providing essential trophic factors to neurons, there are major gaps in our understanding of the molecular mechanisms mediating their diverse actions. This proposal employs a highly tractable system, the glutamatergic Drosophila neuromuscular junction (NMJ), to investigate major questions in glial cell biology with exquisite cellular detail. Our preliminary data provides compelling evidence for a role of glial cells in the plasticity of NMJs, regulating an important pathway required for the differentiation of synapses, and sculpting synaptic connections by a process of pruning. In this project we will (Aim 1) test the hypothesis that glial cells play a primary role in the extension and retraction of synaptic boutons during NMJ expansion, (Aim 2) test the hypothesis that glial cells regulate a Wnt pathway at the NMJ and that this regulation is essential for NMJ development, and (Aim 3) determine the role of the Draper signaling pathway in sculpting synaptic connections at the NMJ. We expect that these studies will provide fundamental insights into the cellular interactions between synapses and glia in live animals and unravel molecular mechanisms by which glia actively sculpt synaptic connections. Project Narrative Glial cells, the major cell type in the human brain, have emerged as important regulators of brain development and physiology, and a number of devastating neurological diseases, such as multiple sclerosis or glioma, are associated with glial dysfunction. This proposal will explore how glia (in live animals) modulate the formation and modification of synapses, the basic functional units through which neurons communicate with other cell types. Our work will provide fundamental knowledge regarding how neurons and glia communicate during the modification of synapses, and is expected to provide important insights into how glial dysfunction might cause disease.